Continuity mistake: The size of the Spartan's shield changes from the action battle sequences (smaller) to the scenes where they are more stationary, as well as, during the arrow attack (larger), presumably allowing greater mobility while fighting. This was confirmed in the Director's Commentary.
Continuity mistake: When Leonidas, and presumably all the other Spartans, use their spears to clear their shields of arrows after the arrow shower from Persian archers, the tips of the arrows are still visibly left on all their shields. In the next shot, when the Spartans regroup for the Persian cavalry charge, the arrow tips are gone. (Granted, this is a highly stylized movie based on a comic book, but that does not change the fact that this is a continuity error.)
Continuity mistake: When the Spartans push off the last people of the the first Persian rush into the water, the shot changes to the Spartans left and we see the Persians falling off the cliff. The sun is bright and behind the falling Persians. After all the Persians have fallen off the Spartans turn their backs to the sun to see arrows being shot at them. As the camera pans up to follow the arrows, the sun is now in front of the Spartans, thus "blotting" out the sun.
Continuity mistake: After Leonidas backs what's left of the first wave of Persians off the cliff, he is seen holding his sword up angrily in his right hand. Then, as the Spartans look up and see the arrows being fired, Leonidas' right hand is now suddenly empty as he walks over to another Spartan and takes from him a spare spear that the Spartan just happen to be holding.
Continuity mistake: Near the end of the movie, just before the final battle scene, Leonidas drops his weapons and shield to the ground. He was holding the shield in the usual manner, and pushes it away to his left. When it hits the ground however, it is suddenly reversed and falls nearby with the front of the shield upward.
Revealing mistake: In the first major battle between the 300 Spartans and the Persians, Leonidas is slaughtering the Persians and the speed of the scene goes from slow motion to fast motion, you see very realistic CGI blood. When the blood hits the the ground it suddenly disappears. This happens many times during this long and awesome scene.
Continuity mistake: Just before Astinos' death, immediately after his father calls to him saying "Astinos, my son", throughout the following shot of Astinos, the horse and rider can be seen in the dust, midway between the ill-fated young man and the right-hand side of the screen, stationary and waiting for his cue. Only after the cut-back to the Captain where he nods in approval is the rider then seen in motion riding towards his target.
Visible crew/equipment: When the camera is focused on an actors face, most of the time you can see white screens and stage lights in their eyes.
Revealing mistake: When the baby is being "inspected" in the beginning, it is pretty obvious that in some of the wideshots that it is just a dummy and not the real baby.
Continuity mistake: After the little boy in Leonidas's arms tells his story about the town being ransacked, he rests his head on Leonidas' shoulder. In the next, more far away shot, his head is no longer resting against Leonidas' shoulder. The transition between the shots was too quick for him to have moved his head.
Visible crew/equipment: Toward the end of the film, the 300 are in a shell style formation - Leonidas is in front, right before he removes his helmet, drops his spear and shield. It is obvious that the sun is shining on Leonidas and the 300 from behind, toward the ocean. But if you look into Leonidas' eyes you'll see white reflections - these are from reflector screens.